ProjectSummary ThisproposalaimstotestthehypothesisthattypeIIafferentsserveascochlearnociceptors. Takingcuesfromthehumancomplaintofhyperacusisafterhearingloss,wewillexaminethestructure andfunctionoftypeIIafferentsinnormalandpost?traumacochleas. Theworkinghypothesisisthat painfulhyperacusis,noxacusis,includeshyperactivityoftypeIIafferents,byanalogytohyperalgesiaof somaticnociceptiveC?fibers. ThuswewillexaminetypeIIstructureandfunctioninnormalandpost?trauma cochleasofratsandmice.Inparallelwewillinvestigatethepropertiesofsurvivingouterhaircellsinpost? traumacochleas.Ourmethodsinclude:exvivoelectrophysiology,lightandelectronmicroscopy,utilization ofoptogeneticandchemogenetictools,andvalidationandquantificationofmousemodelsinwhichtypeII specificbio?markersareexpressed. Anecessaryfirststepistoextendourexvivoexperimentalapproachtooldercochleassothat changeswroughtbyacoustictraumacanbecomparedtothenormalcondition.Wewillcompare damagingsound,ototoxicantibioticsandgeneticallyencodedbiotoxinstoproduceexperimentally tractableeffectsontissueforexvivoexperiments.ThepropertiesandsynapticconnectionsoftypeII afferentsandouterhaircellswillbeexaminedintheexcisedcochleartissueoftheseanimals.Wewill continuetoexploretypeIIspecificgeneticmousemodels.Genetically?encodedreporterproteins, voltage?andcalcium?sensitiveindicators,biotoxins,andopto?andchemo?geneticmodulatorshave becomehighlyinformativetoolsinneurobiologygenerallyandfortheinnerearspecifically.Our ongoingworkhascharacterizedonemouseline,tyrosinehydroxylasepromoterdrivenCre?recombinase expression.ThreeothercandidatetypeIIspecificCrelineswillbevalidatedandquantified.Withsuch transgenicmodelsitbecomespossibletostudyinnervationpatternsbyexpressionoffluorescent reporterproteins,andtoactivate,eliminate,ormodulatetypeIIactivityforanatomicalandphysiological studies.Cre?dependentexpressionofgenetically?modifiedG?protein?coupledreceptors(DREADDS)will providemiceinwhichtypeIIactivitycanbeincreasedordecreasedbyinjectionofanovelsynthetic ligand,dependingonthespecificconstruct.Varyingcombinationsofsystemicandroundwindowdrug deliverywillbeemployedtoincreasethespecificityofexperimentalmanipulations. Theover?archinggoalofthisprogramofexperimentsistocompletethedescriptionoftypeII afferents,astill?unresolvedcomponentofcochlearinnervation. Theworkinghypothesisisthatthese serveascochlearnociceptors. Ifcorrectthesearealikelyneurobiologicalsubstratefornoxacusis (painfulhyperacusis). BydefiningthebasiccellularandmolecularmechanismsoftypeIIfunctionand plasticity,futuretherapeutictargetscanbeidentifiedtoameliorateorpreventnoxacusis.